The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Overview of the Bacterium and Clinical Features of Anthrax:
The causative agent of anthrax is the spore-forming, relatively large (1.0-1.2×3-5 μm), Gram-positive bacillus named Bacillus anthracis. This bacterium is a major bioterrorism threat because its spores are extremely stable, are easily disseminated and are infectious via aerosol. The bacterium forms stable spores in unfavorable environments such as nutrient depletion. B. anthracis has a biphasic life cycle—it can exist as a metabolically inactive endospore or as a rapidly proliferating vegetative cell. The spores are in the range of 1 to 5 μm in diameter, an ideal size for inhalation into alveolar spaces. Once inside the host, the bacterium has both intracellular and extracellular stages of growth. The spores are taken up by macrophages into an acidified endosome, where they germinate and escape the antimicrobial environment.
Outbreaks of this zoonotic disease date back to antiquity and may have been responsible for the fifth and sixth plagues of Egypt described in the Bible. Today the disease is endemic among animals. Robert Koch first traced the complete life cycle of B. anthracis and showed that spores remain viable even in adverse environments. He cultured the bacillus in vitro and inoculated healthy animals that ultimately developed infection.
Anthrax is a serious disease that can affect both animals and humans. People can get anthrax from contact with infected animals, wool, meat, or hides. Anthrax is not known to spread from one person to another. Humans can become infected with anthrax by handling products from infected animals or by breathing in anthrax spores from infected animal products (like wool, for example). People also can become infected with gastrointestinal anthrax by eating undercooked meat from infected animals.
The clinical form of the disease is dependent upon the route of exposure and can manifest as cutaneous, gastrointestinal or inhalational anthrax:
(a) Cutaneous Anthrax: This is the most common form with close to 20% of these cases fatal if untreated. In this form, anthrax is a skin disease that causes skin ulcers and usually fever and fatigue.
(b) Gastrointestinal Anthrax: This form of the disease can result from eating raw/undercooked infected meat. Symptoms include fever, nausea, vomiting, sore throat, abdominal pain and swelling and swollen lymph glands. This form of the disease can lead to blood poisoning, shock and death.
(c) Inhalation Anthrax: This is a serious and often fatal form of anthrax that requires hospitalization. It occurs when the bacillus is inhaled. The initial symptoms may include a sore throat, mild fever and muscle aches. However, within days these symptoms are followed by severe breathing problems, shock, and often meningitis. This form of anthrax requires aggressive antibiotic treatment.
There are currently three known strains of B. anthracis: 
(a) Ames Strain: This strain contains two virulence plasmids which separately encode for the three-protein toxin which mediates the lethal action of anthrax and a poly-glutamic acid capsule which protects the anthrax bacteria from phagocytosis by neutrophils.
(b) Vollum Strain: This strain was initially isolated from a cow in Oxfordshire, UK in 1935. It was used by the British during World War II, and by both the US and UK during the 1960's. It is much more virulent than the Ames Strain. In 1951, Vollum 1B strain was isolated from a scientist who died at Fort Detrick Biological Warfare Center (operated by the US Army). Dr. William A Boyles, a 46 year old scientist, was accidentally exposed to this strain which is more virulent than the original Vollum Strain.
(c) Sterne Strain: This is an attenuated strain which contains the anthrax toxin but not the poly-glutamic acid capsule virulence plasmid.
Conventional Threat to the Civilian Population:
During the first half of the 20th century (up to and including World War II), anthrax has killed hundreds of thousands of animals and human beings in Asia, Australia, Africa, North America and Europe. Exposure to the dormant endospores of the bacterial organism (through the cutaneous, gastrointestinal or respiratory routes discussed above), can result in a fulminant rapidly progressive syndrome ending in septic shock and death in a matter of days. The endospores can produce infections for up to 100 years. Individuals at risk include those occupations which bring human beings into contact with the spores which contaminate animal hides or fur (woolsorters, drum makers), or individuals who eat the meat of infected animals (1).
Bioterrorist Threat to Civilian and Military Populations:
Anthrax also can be used as a weapon. This happened in the US in 2001 when anthrax was deliberately spread through the US postal system by sending letters with powder containing anthrax. This caused 22 cases of anthrax infection. According to a 1970 study by the WHO, the aerosolization of 50 kg of dried B. anthracis spores over a city with a population of 500,000 would incapacitate 125,000 people and kill 95,000, overwhelming medical resources and disrupting the infrastructure of most cities. As a result of programs designed to eradicate anthrax through animal vaccination, sterilization of animal products, only a few cases are now reported each year in the US. The major threat for exposure in the US is through biological warfare and terrorist activity (1).
Classic Examples of the Use of Anthrax in Biological Warfare and Terrorism:
As summarized below, the use of anthrax in biological warfare or as a weapon by terrorists can be catastrophic (1). The development of counter measures against this threat remains an unsolved problem until today (1). There are numerous examples of the use of anthrax endospores for biological warfare (1). Some are highlighted here:
1916: The German Army General Staff provided Swedish fighters with anthrax endospores for use in Finland against the Imperial Russian Army during World War I.
1930-1940: The Japanese Army tested the effect of direct administration of anthrax to human prisoners of war in Manchuria, killing thousands.
1942: The release of anthrax endospores on the Gruinard Island in Scotland by the British Biological Weapons testing program during World War II made that region uninhabitable until very recently.
1940-1945: During World War II, the British Royal Airforce was planning to drop on Germany up to 5 million cow cakes which had been impregnated with anthrax endospores.
1978: The Rhodesian Government used anthrax as a weapon in its war against black nationalists.
1979: Seven years after the USA and the USSR signed the Biological Weapons Convention, which provided for the destruction of all stores of biological weapons, an accidental release (the Sverdlovsk Accident) of anthrax endospores from a biological warfare production facility outside of Moscow killed 68 of the 94 individuals exposed.2001: Attacks against government buildings in the USA (using envelopes which were loaded with anthrax endospores derived from the Ames strain and sent through the mail) occurred. The mortality was low due to the poor quality of the manufacturing process used.
Molecular Mechanism by which Anthrax Kills:
The anthrax bacterium has two virulence factors (1):
(1) A plasmid encoding poly-D-glutamic acid capsule protein which prevents phagocytosis by neutrophils;
(2) A plasmid which encodes the following three proteins which together constitute the anthrax Toxin: the protective antigen (PA), the edema factor (EF), and the lethal factor (LF).
The PA is non-toxic by itself, but the carboxyterminal end of this 83 kDa protein binds to two cellular receptors: the anthrax toxin receptor and the capillary morphogenesis protein 2 receptor. Neither LF nor EF is able to bind to or enter mammalian cells by themselves, but do so once bound to PA. EF is a calmodulin dependent adenylate cyclase. LF is a zinc dependent metalloprotease (1). The 83 kDa PA released by the anthrax bacterium binds to its cellular receptors. PA is then cleaved by a furin cellular protease into a 20 kDa fragment (at the N-terminus) and a 63 kDa fragment (at the C-terminus). The removal of the 20 kDa fragment reduces steric hindrance which otherwise prevents oligomerization of the PA into a heptameric ring-shaped structure. This heptameric structure can bind three molecules (any mixture of LF or EF) at nanomolar concentrations. This structure then relocates to detergent-resistant lipid microdomains in the plasma membrane where the heptamer of PA bound to EF and LF is internalized into the cell by endocytosis. Acidification within the endosome then allows the heptamer to insert itself into the endosomal membrane where it then translocates the EF and LF into the cytoplasm (1). LF then leads to cell death in host tissues and monocytes through inhibition of the MEK. LF has a zinc dependent metalloprotease action. This suppresses the function of neutrophils and monocytes. EF is a calmodulin dependent adenylate cyclase (1). The EF induced increase in adenylate cyclase may result in degranulation of monocytes (1). The complex formed between calmodulin and adenylate cyclase blocks calcium dependent signaling essential to the immune response. LF and EF target the endothelial cells that line vessels and serous cavities (pericardial cavity, pleural cavity and the peritoneal cavity), causing vascular leak, hypovolemic shock, septemic shock, and cell death (1).
Previous Work on Vaccine Strategies for Anthrax:
Louis Pasteur produced the first veterinary live cell vaccine for anthrax in 1881, which comprised B. anthracis attenuated by passage at elevated incubation temperature. He tested the effect of the injection of anthrax into two groups of sheep, one group vaccinated 30 days earlier (two injections at 15 day intervals) and one not vaccinated with his vaccine. All the animals not vaccinated died and all of the animals in the vaccinated group lived (1).
The first human vaccine was introduced in 1954 (cell free). There are two types of vaccines currently available:
(a) Live-Attenuated Vaccine: This is called the Russian-Georgian vaccine (STI) which has been derived from the Stern Strain. This vaccine is not considered safe for use in any but the most fit human subjects due to the severity of the side effects.
(b) Cell Free Vaccine: An example is the USA vaccine (1) which is manufactured by Emergent BiSolutions (BioThrax) which is adsorbed onto aluminum hydroxide as adjuvant. This vaccine is approved by the US FDA for administration at 0, 2, and 4 weeks initially and then at 6, 12, and 18 months and yearly thereafter, for military personnel who are entering areas known to be endemic for anthrax. The week 2 dose was made optional in 2008. According to information on line in Google as part of the Anthrax Vaccine Immunization Program (AVIP), this vaccine is based on a fragment of the “protective antigen” (PA) which is described in the preceding section (1). This vaccine is designed to induce antibodies against the PA so as to neutralize the ability of the Bacillus anthracis to cause disease.
Shortcomings of Current Vaccine Strategies:
Some of the shortfalls of currently employed vaccines are discussed below.
Low Potency: As outlined above, there are three targets through which the lethality of the anthrax toxin could be blocked by neutralizing antibodies: the PA, the LF and the EF. The currently-employed USA vaccine, which is based on recombinant PA, appears to have a low potency and limited memory since it requires the use of an adjuvant and multiple boosting every 6 months×3 following induction and yearly thereafter (1). This vaccination schedule suggests that the vaccine itself is not very potent and requires constant boosting to maintain protection due to a weak memory response.Toxicity: Press accounts of short and long term toxicities of this vaccine are available in the public domain. The side effects that have been associated with this vaccine (Morbidity and Mortality Weekly Report 59: page 11, 2009) include 10% serious adverse events (deaths, hospitalizations and permanent disability). In addition, short term adverse events have been observed at the injection site such as erythema, pain, itching and nodules. In addition, systemic symptoms such as fever, chills, myalgia, arthralgia and malaise are also associated with this vaccine.
Given the rise of virulent strains of B. anthracis which display antibiotic resistance, the goal of development of anthrax vaccines for high risk populations has emerged as an important priority. Unfortunately, this goal has not yet been realized. One factor that could prevent the success of vaccination is that the patients who are admitted to hospitals are often of advanced chronological age, are debilitated and/or are immunosuppressed as a result of chronic disease (4-7). These patients often do not respond to vaccination due to the diminished expression of CD40L in the CD40L helper T cells of these people (8-9). In this regard, another serious issue is that passive immunotherapy with opsonizing antibodies is unable to completely protect these individuals against anthrax.